PROJECT SUMMARY/ABSTRACT Tissue-resident memory T (TRM) cells are a subset of memory T cells that resides in tissue without recirculating through the bloodstream and are phenotypically, functionally and transcriptionally distinct from circulating memory T cells. CD8 TRM cells act as first responders to pathogen re-encounter and mediate effective protective immunity by triggering rapid innate and adaptive immune responses through cytokine production. However, the mechanisms regulating CD8 TRM cell development are still not completely understood. CD8 TRM cell development involves several critical aspects including 1) T cell priming in draining lymphoid organs, 2) T cell migration into non-lymphoid tissues, 3) in situ TRM cell differentiation and 4) TRM cell maintenance. The intestinal mucosa is constantly exposed to commensal bacteria and potential pathogenic microbes and is continuously surveilled by CD8 TRM cells emphasizing the importance of understanding CD8 TRM cell development in the intestine. It's generally thought that priming at specific lymphoid organs dictates the migration patterns of effector CD8 T cells into specific non-lymphoid tissue while factors existing in the local environment such as cognate antigen and inflammation determine in situ TRM cell formation and maintenance. However, comparing different routes of immunization using a recombinant Listeria monocytogenes strain containing a mutation in the internalin A protein (InlAM rLM) that allows efficient invasion of the murine intestinal epithelium, we demonstrate that priming in the mesenteric lymph nodes (MLN) is critical for in situ CD8 TRM cell differentiation in the intestine. However, many questions remain regarding this process and mechanistic insights are lacking for how MLN priming regulates these processes. The overall hypothesis to be tested is that MLN priming licenses CD8 TRM cell development and protective immunity in the intestine. The research goals will be accomplished in three specific aims. Specific Aim 1: Determine the impact of immunization route on CD8 TRM cell development. Specific Aim 2: Determine the impact of priming location on CD8 TRM cell-mediated protective immunity. Our goal of the above proposed study is to understand mechanisms regulating CD8 TRM cell development and function, which is highly important and significant with regard to CD8 TRM cell-mediated protective immunity at mucosal surface and will help us to develop successful vaccination strategies. Information generated in this exploratory proposal will provide critical new insights, change the thinking about intestinal CD8 TRM cell development, and lay the foundation to greatly advance the field.